Esters of bile acid degradation products and a process for preparing same



United States Patent 3,07d,i iltl ESTERS 0F BEE AlClD DEGRADATEON PEQD- UCTS AND A PRES FUR PREPARING SAME Ha ry 6. Penn, 2678 Glendower Ave, Los Angeles, Calif. No Drawing. Fi ed Aug. 15, 1957, Ser. No. 67$,53ll Claims. (Ci. 26il397.1)

This invention relates to lower alkyl esters of bile acid degradation products having utility as testing agents in serological diagnosis of cancer and to a process for the preparation of such esters. Thi application is a continuation-in-part of my copending application Serial Number 451,284, filed August 20, 1954, now abandoned, which in turn is a continuation-impart of my application Serial Number 368,009, filed July 14, 1953, now abandoned.

The effectiveness of cancer treatments depends to a great extent upon the early diagnosis of the disease. Accurate diagnosis of cancer in the very early stages, before any visible symptoms of the disease have appeared, is a very difiicult matter. A positive test for cancer in its early stages would, therefore, be of great value in the successful combating of the disease.

Accordingly, an object of the invention is to provide serologically active agents having application .in a test for cancer and processes for the production of such agents.

The above and other objects will become apparent after reading the following specification.

Generally stated, the scrological test comprises treating blood serum from a person or other animal with an agent which will give an accurate indication as to whether or not the donor of the serum has cancer. It has been discovered that certain bile acid degradation products and esters of these products, when mixed under standardized conditions with a serum obtained from a person having a malignant new growth, will produce a characteristic flocculation reaction not observed when the serum is from a person not having a malignant growth. By degradation products of the bile acids and their esters are meant products produced by such reactions as hydrolysis, reduction, dehydrogenation, oxidation, pyrolysis, dehydration, decarboxylation, and other reactions acting on the original bile acids or bile acid esters, but not destroying the ring structure of the cholane nucleus of the original acids or esters.

It has been found that the bile acid degradation product preferably is a degradation product of a bile acid such as cholic acid, desoxycholic acid, chenodesoxycholic acid and lithocholic acid, and particularly an ester of such degradation product. More specifically stated, it has been found that the esters of cholenic acids, of choladienic acids and of cholatrienic acids may be used as serological cancer test agents as described above.

Of these esters, the lower alkyl esters, such as the methyl and the ethyl esters, are readily made in high yields from the bile acids by dehydrating the esterified acid, and are desirable cancer test agents for this reason. Among the desirable test agents of the group are the esters of desoxycholenic acid, desoxycholadienic acid, and desoxycholatrienic acid.

Included among the cancer test agents for use in the serological method are partially dehydrated cholic acid, desoxycholic acid, chenodesoxycholic acid and lithocholic acid and their esters. That is, one or both of the hydroxyl groups in the molecule may be removed from the cholic acid nucleus and only one of the hydroxyl groups may be removed from the desoxycholic acid and the chenodesoxycholic acid nuclei to give dihydroxy cholenic, hydroxy choladienic and hydroxy cholenic 3,7fi,iili Patented Jan. 29, 193

acids and esters respectively. Apocholic acid and its esters, for example, are included among these cancer test agents. One or both of the hydroxyl groups, as the case may be, of these hydroxy cholenic nuclei may be substituted by such radicals as halogen, sulfur, alkoxy, al-kyl, substituted alkyl, amino, cyanogen, aldehyde, carboxyl, ester, amide, ketone, nitro, and sulfonic radicals for example. It has been found that the derivatives produced by substituting the hydroxyl radical in the 3-position of the hydroxy cholenic nucleus by a halogen atom, particularly the substitution of a chlorine atom for the hydroxyl in the 3- position of an ethyl ester of a 3-hydroxy cholenic acid, are relatively stable "crystallin substances.

The cancer test agents employed in the serological test can be prepared by any one of several methods. The bile acid or its ester can be pyrolized or treated with a dehydrating agent to introduce one or more double bonds into the acid or ester molecule and thus produce the desired cholenic acid or ester test agent. In a preferred method, the bile acid dissolved in an anhydrous alcohol is dehydrated with concentrated sulfuric acid amid refrigeration in an inert atmosphere. The resulting reaction mixture is added to cold water and the crude cholenic acid ester is extracted from this aqueous mixture with an organic solvent. The crude ester can-be puritied by any suitable method such .as by chromatographic fractionation.

Alternatively, the hydroxyl groups of the ester of the bile acid can be acetylated, or esterified, and the resulting acetyl derivative can be pyrolyzed in an inert atmosphere. The pyrolysis product, containing the cholenic acid ester can be purified by methods such as vacuum distillation and chromatographic fractionation.

Higher esters of cholenic acids can be prepared by treating the cholenic acid with phosphorous pentahalide to form the acid halide. The halide is then reacted with the higher alcohol to form the higher ester of cholenic acid. The crude ester can be purified by the methods suggested above.

Other methods of preparing the cancer test agents can be used. The above methods are outlined by Way of illustration only and not by Way of limitation. The following examples are given to additionally illustrate, in more detail certain specific methods of preparing certain test agents and of performing a specific embodiment of the cancer test.

Example 1 Desoxycholic acid of GP. grade preferably is purified by the method of Reichstein and Sorldn, as described in Helv. Chim. Acta, vol. 25, page 797, 1942. A lO-gram portion of the purified acid is dissolved in 50 grams of absolute ethyl alcohol in a 500 ml. three-neck flask. This 5:1 ratio of alcohol to desoxycholic acid has been found 'to b'e very satisfactory and is maintained in the preparation of the derivative in any desired quantity. Mild application of heat on the steam bath may be necessary to facilitate rapid solution. The flask is then placedin an icebath. A glass stirring rod, attached to a stirring motor, is placed in the middle neck of the flask and nitrogen gas is passed over the reaction mixture. A separatory funnel containing 100 ml. of cold concentrated sulfuric acid at -l'0 C. is placed in one of the side necks, and a thermometer in the other. The weight ratio of 90% sulfuric acid to desoxycholic acid should be maintained "at about 10:1 to obtain a maximum yield of product.

The reaction is initiated by adding sulfuric acid drop by drop. The temperature is immediately brought up to about 35 'C. and is maintained between about 35 to 40 C. throughout the reaction. The rate of addition of the sulfuric acid should be as rapidly as possible at the maintained reaction temperature and, takes from 8 to 10 minutes. Stirring is continued for one to two minutes after all the sulfuric acid is added. The color of the reaction mixture proceeds from a colorless transparent to a yellow-orange solution. The resulting reaction mixture is placed in a refrigerator maintained at about 4 C. to about 10 C. for about 18 to about 24 hours.

The cold reaction mixture is added to approximately 1 liter of ice-cold distilled water in a 2-liter separatory funnel and the resulting emulson is extracted with approximately 600 to 800 ml. of pure n-hexane. The n-hcxane extract is thoroughly washed with several portions of distilled water until all the brownish precipitate is removed. The pH of the aqueous wash should be that of the distilled water before proceeding to the next step. The n-hexane extract is then washed with several portions of 5% sodium carbonate solution. An emulsion and yellowish-white precipitate (sodium salts of the unreacted bile acid) are formed with the first aqueous carbonate wash. Most of the precipitate is removed by two to three 150 ml. aqueous carbonate washes with vigorous shaking. Wash with 5% sodium carbonate solution is discontinued when the carbonate solution remains clear upon its addition to the hexane extract indicating that all the unreacted bile acid has been washed from the extract. The latter is finally washed with distilled water until neutral to nitrazine paper.

The washed hexane extract is then dried over anhydrous sodium sulfate for to 30 minutes and the hexane is removed from the extract by steam distillation. The residual yellow viscous oil is dried under vacuum. The yield of crude product usually obtained is approximately 7.0 to 8.5 grams of this oil.

The crude product is dissolved in about 75 to 100 ml. of pure n-hexane. This solution is percolated chromatographically through a column of activated alumina, using about 30 to about 40 gm. of the alumina per gm. of dissolved crude product. The chromatogram is developed in the column by eluting the same with an additional 300 to 325 ml. of n-hexane. The following bands are observed in the column under fluorescent light when reading from top to bottom on the column:

(A) Deep olive zone (B) A blue-green zone (C) A purple-blue fluorescent zone (D) Two narrow blue fluorescent zones close together near the bottom of the column.

Elution of the column with n-hexane is continued until the D material enters the filtrate. Receiving flasks are then changed and elution is continued with about 2.0 to 2.5 liters of n-hexane.

The second filtrate appears as a clear, transparent, purplish-tinted material under fluorescent light. This filtrate contains the desired cancer test agent. The solvent is removed from this fraction and a residue of about 1.0 to 2.5 grams is obtained. The residue is redissolved in n-hexane, and rechromatographed, using gm. of activated alumina per gm. of redissolved material. The column chromatogram is then developed with n-hexane and examined under ultraviolet light. The column should, however, not be exposed excessively to ultraviolet light. The blue fluorescent zone, if present, is permitted to enter the filtrate. Receiving flasks are then changed and the purple fluorescing fraction is collected separately. This fraction contains the desired cancer test material. Most of it is obtained in the first 300 to 400 ml. of the second filtrate.

The solvent is removed from this fraction by steam distillation at low pressure. The residual, clear, colorless oily product is dried under vacuum and dissolved in pure 95% ethanol in the ratio of 20 mg. product per ml. ethanol. The resulting solution of ethyl ester of choladienic acid test agent is stored in brown bottles under nitrogen.

In the above example the desoxycholic acid in absolute alcohol solution is ultimately converted to an ethyl ester of choladienic acid. The concentrated sulfuric acid appears to convert the bile acid to the ester and to remove two molecules of water from the bile acid molecule, corresponding to the two hydroxyl groups in the bile acid nucleus. Two double bonds are thereby introduced into the cholane nucleus. An intermediate lactone is believed first to be produced, but later evidently is converted to product ethyl ester of choladienic acid. Double bond isomerism involves the number 2 carbon atom so that the double bond can occur at the 2,3-position as well as at the 3,4-position. The final product is a mixture of at least these isomers.

Although the cancer test agent, when added to blood serum, will indicate the presence of cancer in the donor of the serum in various ways, it has been found convenient, when using certain of the cancer test agents, such as the esters of cholenic acids for example, to base the test on the character and presence or absence of flocculation or turbidity produced in the mixture of serum and cholenic acid ester. It is to be understood, however, that the invention comprehends the observance of other physical or chemical phenomena exhibited on addition of the test agent to the serum, and is not restricted to flocculation observance.

Other synthetically developed esters of choladienic acid are useful in the seroflocculation reaction. Tests appear to indicate that the lower molecular weight esters are more satisfactory than those of relatively higher molecular weight. Methyl or ethyl esters of choladienic acid, appear better suited to the test than the capryl or isoamyl esters, for example. The following examples illustrate methods of preparing various ester forms of the cancer test agent for use in the serological procedure described.

Example 2 A solution of 3 gm. of choladienic acid in ml. of methanol containing 1 ml. of 90% concentrated sulfuric acid was refluxed for 3% hours. The reaction mixture was poured into about 700 ml. of water and the precipitated oil was extracted with ethyl ether. The other solution was washed with water, 10% sodium carbonate solution, with water to neutrality, and dried over sodium sulfate. The filtered other solution yielded an oil residue on evaporation of the ether. The residue was dissolved in ml. of methanol, filtered, and allowed to stand at room temperature until crystallization began and then placed in a refrigerator to complete the crystallization. 2.21 gm. of product methyl ester of choladienic acid was obtained which after one recrystallization from methanol melted at 80.5-81 C.

The ethyl ester of choladienic acid is similarly prepared from choladienic acid and ethanol. Both esters gave satisfactory seroflocculation reactions.

Example 3 Another method of preparation of the methyl ester of choladienic acid is as follows: A solution of 50 gm. of desoxycholic acid in 200 ml. of methanol containing 1 ml. of 90% concentrated sulfuric acid was allowed to stand at room temperature for 24 hours. The solution was poured into a mixture of ice and water. An oil separated which crystallized after standing for a short time. Sodium carbonate was added until the solution was alkaline to litmus and the solid was filtered. The rather lumpy precipitate was ground in a mortar, washed with water and dried in a vacuum desiccator. The solid was then recrystallized from dilute methanol and gave 50.5 gm. of product methyl ester of desoxycholic acid melting at 85-88 C.

A solution of 50 gm. of desoxycholic acid methyl ester in a solution of 30 ml. of dry pyridine and 50 ml. of acetic anhydride was heated on a steam bath for '22 hours and then refluxed for two hours. The reaction mixture was poured into ice water and allowed to stand until the oil began to solidify. The supernatant liquid was decanted and the semisolid residue was washed with fresh amounts of cold water until it was completely solid. The solid was dissolved in ethyl .ether and filtered. The ether solution was washed twice with dilute hydrochloric acid, then with water to neutrality, and dried over calcium chloride for about one hour. The dried solution was evaporated on a steam bath and the residual 'oil was dissolved in approximately 3 volumes or methanol. The derivative crystallized from the methanol on standing yielded 43.5 gm. of product methyl diacetyldesoxycholate, melting at ;1l8120 C. A second crop of gm. of crystalline product was obtained from the mother liquid. The total Purified desoxycholic acid is heated for 1 hour at 250 to 280 C. under vacuum (10-4 microns) over nitrogen gas. The residual oil (page yellow color) is distilled at 315 to 350 C. for 2 hours at 0.001 mm. pressure. A crystal clear distillate is obtained in the receiving flask. This is dissolved in peroxide free ethyl ether (treated with FeSO CaOH and water). The ethyl ether layer is extracted with N KOH several times and then with water to neutrality. The aqueous KOH and water extracts are combined and washed with ether 3 times. The ether layer is set aside (contains the neutral pyrolytic fraction).

The washed aqueous KOH phase is acidified with 4 N HCl. The resulting precipitate is dissolved in ether. The resulting ether solution is washed to neutrality with water and dried over anhydrous sodium sulfate for several hours. The dried ether solution is evaporated by steam distillation and the residual clear viscous oil is dried thoroughly under vacuum. The oil is crystallized from absolute methyl alcohol. The white crystals formed are filtered and washed once with cold methyl alcohol. This precipitate is then dried under vacuum. M.P. of the white crystals of choladienic acid is 132-133 C.

The choladienic acid is dissolved in either ethyl alcohol or methyl alcohol-depending on whether a methyl or ethyl ester is desired-using 1 part dienic acid to 10 parts alcohol containing a few ml. of 90% concentrated sulfuric acid. This solution is kept at room temperature overnight. After 24 hours distilled water is added to dilute the alcoholic solution to approximately 70% concentration. Barium carbonate in sufiicient quantity is added to remove the sulfate ions.

The alcohol is removed by distillation under reduced pressure. The residual aqueous phase is extracted with peroxide free ethyl ether 3 times. This ether phase is washed till neutral and then extracted wit-h N sodium carbonate several times. The ether layer is then washed till neutral and dried over anhydrous sodium sulfate for several hours.

The ether is removed by distillation and the oil dried under vacuum. The oil is distilled under high vacuum (0-10 microns) at 235 to 250 C. The clear distillate is crystallized from absolute methyl alcohol 2 times. The second crystallization is carried out overnight at 4 C. The flaky white crystals are filtered in a cold room (0 0.), collected, and dried under vacuum. The M.P. of the ethyl ester of choladienic acid prepared as above is 6 38 'C. and that of the methyl ester of "choladienic acid is to C. It "gives satisfactory seroflocculation reactions.

Example 5 The ethyl ester of choladienic acid was prepared by th'eesterification of choladienic acid with absolute ethanol and sulfuric acid by the method of Example 3. The product could not be made to crystallize by ordinary methods and was, therefore, chromatographed on alumina. 1.5 vgm. of a colorless oil was obtained which still could not be crystallized. 'It was distilled in vacuum and boiled at 214-215 C. at 1 mm. pressure. The distillate was crystallized from methanol and melted at 45.5 to 49 C. It gave serofiocculation reactions.

Example '6 The capryl ester of choladienic acid was prepared in the following manner:

To 1 gm. of choladienic acid in 10 ml. of dry chlorovform 0.6 .gm. of phosphorus pentachloride was added. When the reaction was complete, 1 ml. of capryl alcohol was added to the reaction mixture. The solution was allowed to remain at room temperature for about an hour, and was then evaporated on a steam bath to a volume of about 5 ml. Ethyl ether was added thereto and the resulting solution was extracted twice with water, once with dilute sodium carbonate solution and then with water to neutrality. The resulting ether solution was again washed with dilute potassium hydroxide solution and again with water to neutrality. The oily residue obtained on removal of the ether did not crystallize. It

was chromatographed on '30 gm. of alumina and gave 820mg. of oily product. It gave serofiocculation reactions.

Example 7 The isoamyl ester of choladienic acid was prepared in the same manner as the capryl ester. From 1 gm. of ch'oladi'enic acid 818 mgm. of ester was obtained after chromatography on 30 gm. of alumina.

Example 8 The normal butyl ester was prepared from 1 gm. of 'choladienic acid, 50 ml. of normal butyl alcohol and 1 ml. of concentrated sulfuric acid by the method of Ex ample 3. The ester could not be crystallized even after chromatography on alumina. The 1 gm. of ester obtained was distilled and boiled at 234 C. at 1 mm. pressure. It gave seroflocculating reactions.

The following example illustrates the preparation of the amide of choladienic acid for use as a cancer test agent in the method of the invention.

Example 9 The amide of choladienic acid was prepared by adding 0.3 gm. of phosphorus pentachloride to a solution of /2 gm. of choladienic acid in about 10 m1. of ethyl ether. When the reaction was complete, the solution was poured into concentrated ammonium hydroxide at '5 C. The precipitated solid was filtered and recrystallized from dilute methanol in colorless needles which melted at to 146 C.

The example below illustrates the preparation of a cancer test agent for use in the method of the invention wherein one of the hydroxyl groups of a bile and degradation product is substituted by a chlorine atom.

Example 10 A quantity of 3 a-hydroxy-A -choleic acid was esterified with absolute ethanol essentially according to the method used by L. L. Engel et al., I. Bio. Chem, vol. 162, page 565, year 1946, for the preparation of the methyl ester. The ethyl ester crystallized satisfactorily from purified hydrocanbon solvent as colorless needles.

To a portion of the ethyl ester dissolved in chloroform and cooled to C. was added while stirring the solution a small amount of powdered calcium carbonate and, in small portions, an excess of phosphorous pentachloride. The pentachloride was added during about 20 minutes, and stirring at 0 C. was continued for an additional period of about 100 minutes.

The resulting reaction mixture was poured into a dilute sodium bicarbonate solution at 0 C. This mixture was extracted with ether and the ether extract was washed with distilled water. The ether was evaporated from the extract under reduced pressure and a colorless residual oil resulted. This oil, upon solution in a small amount of methanol and refrigeration for several hours, resulted in a 73% yield of colorless crystals of ethyl 3 B-chloron -cholenate, melting at 73 C.

This compound was found to be stable under ordinary conditions and was found to be a satisfactory seroflocculation agent.

Although the invention is herein shown and described in what is conceived to be the most practical and me ferred embodiment, it is recognized that departures may be made therefrom within the scope of the invention, which is not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent means, test agents, and test methods.

I claim:

1. The ethyl ester of an acid selected from the group consisting of A choladienic acid, A3111 choladienic acid, and B S-halogenated A cholenic acid.

2. Ethyl 3,8-chloro-A cholenate. I

3. A serological test agent comprising a mixture of ethyl A and ethyl A choladienates.

4. A method for obtaining an ethyl ester of choladienic acid, comprising: mixing a solution of desoxycholic acid in anhydrous ethyl alcohol with concentrated sulfuric acid until the relative proportions by weight of parts sulfuric acid to one part desoxycholic acid is obtained while maintaining a reaction temperature between about C to C. to thereby remove the hydroxyl groups from the acid nucleus and to introduce double bonds therein while esterifying the acid side chain, chilling the reaction mixture, diluting with water, extracting the reaction product from the resulting emulsion with an organic solvent, and recovering said ester from said solvent extract.

5. A method for obtaining an ethyl ester of choladienic acid, comprising: adding concentrated sulfuric acid to a solution of desoxycholic acid in about five volumes of absolute ethyl alcohol while maintaining the reaction temperature between about 35 to 40 C. until a weight ratio of sulfuric acid to desoxycholic acid of about 10 to 1 has been reached, cooling the resulting reaction medium and maintaining it at a temperature below about 10 C. for about 18 to 24 hours, diluting the mixture with water, extracting the reaction product from the resulting emulsion with n-hexane, washing and drying the extract, distilling the solvent frorn the extract, redissolving the residue in n-hexane, chromatographically fractionating the n-hexane solution in a column of activated alumina, eluting the column with n-hexane and collecting the fraction appearing purplish-blue under fluorescent light, and separating the choladienic acid ester product from the solvent in said collected fraction.

References Cited in the tile of this patent UNITED STATES PATENTS Kendall Apr. 22, 1947 Bernstein et al Nov. 29, 1955 I logische Chemie, vol. 212, pp. 269-77 (1932).

Wieland et al.: Chem. Abst, Vol. 27 (1933), 511. Yamazaki et al.: Chem. Abst., vol. 48 (1954), 2737 (efifective date 1952). 

1. THE ETHYL ESTER OF AN ACID SELECTED FROM THE GROUP CONSISTING OF $2,11 CHOLADIENIC ACID, $3,11 CHOLADIENIC ACID, AND 3B-HALOGENATED $11 CHOLENIC ACID. 